The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be, or to describe, prior art to the invention. All publications are incorporated by reference in their entirety.
Free phosphorus and phosphonic acids and their salts are highly charged at physiological pH and therefore frequently exhibit poor oral bioavailability, poor cell penetration and limited tissue distribution (e.g. CNS). In addition, these acids are also commonly associated with several other properties that hinder their use as drugs, including short plasma half-life due to rapid renal clearance, as well as toxicities (e.g. renal, gastrointestinal, etc.) (e.g. Antimicrob Agents Chemother May 1998; 42(5): 1146-50). Phosphates have an additional limitation in that they are not stable in plasma as well as most tissues since they undergo rapid hydrolysis via the action of phosphatases (e.g. alkaline phosphatase, nucleotidases). Accordingly, phosphate esters are frequently used as a prodrug strategy, especially for water insoluble compounds, since the phosphate group enables high water solubility and thereby enables delivery of the drug parenterally.
Prodrugs of phosphorus-containing compounds have been sought primarily to improve the limited oral absorption and poor cell penetration. In contrast to carboxylic acid proesters, many phosphonate and phosphate esters fail to hydrolyze in vivo, including simple alkyl esters. The most commonly used prodrug class is the acyloxyalkyl ester, which was first applied to phosphate and phosphonate compounds in 1983 by Farquhar et al. J. Pharm. Sci. 72(3): 324 (1983). The strategy entails cleavage of a carboxylic ester by esterases to generate an unstable hydroxyalkyl intermediate which subsequently breaks down to generate the drug and an aldehyde. In some cases this biproduct (e.g., formaldehyde), can be toxic. This strategy is used to enhance the bioavailability for several drugs. For example, the bis(pivoyloxymethyl) prodrug of the antiviral phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) has been studied clinically for the treatment of CMV infection and the bis(pivaloyloxymethyl) prodrug of the squalene synthetase inhibitor BMS187745 is undergoing clinical evaluation for the treatment of hypercholesterolemia and associated cardiovascular diseases. The marketed antihypertensive, fosinopril, is a phosphinic acid angiotensin converting enzyme inhibitor that requires the use of an isobutryloxyethyl group for oral absorption.
Several other esters have been used as prodrugs of phosphorus-containing compounds. For example, aryl esters, especially phenyl esters, are another prodrug class reported to be useful for the delivery of phosphorus-containing compounds. DeLambert et al., J. Med. Chem. 37: 498 (1994). Phenyl esters containing a carboxylic ester ortho to the phosphate have also been described. Khamnei and Torrence, J. Med. Chem.; 39:4109-4115 (1996).
Benzyl esters are reported to generate the parent phosphonic acid. In some cases using substituents at the ortho- or para-position can accelerate the hydrolysis. Benzyl analogs with an acylated phenol or an alkylated phenol can generate the phenolic compound through the action of enzymes, e.g. esterases, oxidases, etc., which in turn undergoes cleavage at the benzylic C—O bond to generate the phosphonic acid and the potentially toxic quinone methide intermediate. Examples of this class of prodrugs are described by Mitchell et al., J. Chem. Soc. Perkin Trans. I 2345 (1992); Brook, et al. WO 91/12721. Still other benzylic prodrugs have been described containing a carboxylic ester-containing group attached to the benzylic methylene. Glazier et al. WO 91/19721.
Cyclic phosphonate esters have also been described for phosphorus-containing compounds. In some cases, these compounds have been investigated as potential phosphate or phosphonate prodrugs. Hunston et al., J. Med. Chem. 27: 440-444 (1984). The numbering for these cyclic esters is shown below:
The cyclic 2′,2′-difluoro-1′,3′-propane ester is reported to be hydrolytically unstable with rapid generation of the ring-opened monoester. Starrett et al. J. Med. Chem. 37: 1857-1864 (1994).
Cyclic 3′,5′-phosphate esters of araA, araC and thioinosine have been synthesized. Meier et al. J. Med. Chem. 22: 811-815 (1979). These compounds are ring-opened through the action of phosphodiesterases which usually require one negative charge.
Cyclic 1′,3′-propanyl phosphonate and phosphate esters are reported containing a fused aryl ring, i.e. the cyclosaligenyl ester, Meier et al., Bioorg. Med. Chem. Lett. 7: 99-104 (1997). These prodrugs are reported to generate the phosphate by a “controlled, non-enzymatic mechanism[s] at physiological pH according to the designed tandem-reaction in two coupled steps”. The strategy was purportedly used to deliver d4-T monophosphate to CEM cells and CEM cells deficient in thymidine kinase infected with HIV-1 and HIV-2.
Unsubstituted cyclic 1′,3′-propanyl esters of the monophosphates of 5-fluoro-2′-deoxy-uridine (Farquhar et al., J. Med. Chem. 26: 1153 (1983)) and ara-A (Farquhar et al., J. Med. Chem. 28: 1358 (1985)) were prepared but showed no in vivo activity. In addition, cyclic 1′,3′-propanyl esters substituted with a pivaloyloxy methyloxy group at C-1′ was prepared for 5-fluoro-2′-deoxy-uridine monophosphate (5-FdUMP; (Freed et al., Biochem. Pharmac. 38: 3193 (1989); and postulated as potentially useful prodrugs by others (Biller et al., U.S. Pat. No. 5,157,027). In cells, the acyl group of these prodrugs underwent cleavage by esterases to generate an unstable hydroxyl intermediate which rapidly broke down to the free phosphate and acrolein following a β-elimination reaction as well as formaldehyde and pivalic acid.
Cyclic phosphoramidates are known to cleave in vivo by an oxidative mechanism. For example, cyclophosphoramide is thought to undergo oxidation at C-1′ to form the hydroxylated intermediate, which like the 1′-substituted cyclic 1′,3′-propane esters described above, breaks down to acrolein and the corresponding phosphoramidate. Cyclophosphoramidates were also prepared as potential prodrugs of both 5-FdUMP and araAMP and shown to have modest activity in vivo.
A variety of substituted 1′,3′ propanyl cyclic phosphoramidates, wherein 1′ represents the carbon alpha to the nitrogen were prepared as cyclophosphamide analogs (Zon, Progress in Med. Chem. 19, 1205 (1982)). For example, a number of 2′- and 3′-substituted proesters were prepared in order to decrease the propensity of the α,β-unsubstituted carbonyl bi-product to undergo to a Michael reaction. 2′-Substituents included methyl, dimethyl, bromo, trifluoromethyl, chloro, hydroxy, and methoxy whereas a variety of groups were used at the 3′-position including phenyl, methyl, trifluoromethyl, ethyl, propyl, i-propyl, and cyclohexyl. Analogs with a 3′-aryl group underwent oxidation alpha to the nitrogen and accordingly exhibited anticancer activity in the mouse L1210 assay. A variety of 1′-substituted analogs were also prepared. In general these compounds were designed to be “pre-activated” cyclophosphamide analogs that bypass the oxidation step by already existing as a 1′-substituted analog capable of producing the final compound, e.g. hydroperoxide and thioether. A series of 1′-aryl analogs were also prepared in order to enhance the oxidation potential. In contrast to the 1′-hydroperoxy analogs, the 1′-aryl compounds exhibited either no activity or very poor activity in the standard anticancer in vivo screen assay, i.e. the mouse L1210 assay. The lack of activity was postulated to arise from the steric hinderance of the phenyl and therefore the limited oxidation of the prodrug. Support for this postulate was the potent activity of the acyclic phenyl keto analog which exhibited activity similar to cyclophosphamide.
Cyclic esters of phosphorus-containing compounds are reported in the chemical literature, however they were not tested as prodrugs in biological systems. These cyclic esters include:
[1] di and tri esters of phosphoric acids as reported in Nifantyev et al., Phosphorus, Sulfur Silicon and Related Eelements, 113: 1 (1996); Wijnberg et al., EP-180276 A1;
[2] phosphorus (III) acid esters. Kryuchkov et al., Izv. Akad. Nauk SSSR, Ser. Khim. 6: 1244 (1987). Some of the compounds were claimed to be useful for the asymmetric synthesis of L-Dopa precursors. Sylvain et al., DE3512781 A1;
[3] phosphoramidates. Shih et al., Bull. Inst. Chem. Acad. Sin, 41: 9 (1994); Edmundson et al., J. Chem. Res. Synop. 5: 122 (1989); and
[4] phosphonates. Neidlein et al., Heterocycles 35: 1185 (1993).
Numerous phosphorus-containing compounds are known to exhibit pharmacological activity but remain far from optimal due to one or more of the above-described limitations. Some of the activities described include phosphonic acids that are useful as antihypertensives and therapy for heart failure via inhibition of NEP 24.11, phosphonic acids that are useful for treating a variety of CNS conditions (stroke, epilepsy, brain and spinal cord trauma, etc.) via binding to excitory amino acid receptors (e.g. NMDA receptor), bisphosphonic acids that are useful for treating osteoporosis, phosphonic acids that are useful as lipid lowering agents (e.g. squalene synthase inhibitors), phosphonates that are useful in treating inflammation (e.g. collagenase inhibitors), phosphonates and phosphates that are useful in treating diabetes, cancer and parasitic and viral infections.
Phosphates and phosphonates that are known to be particularly useful in glucose lowering activity and therefore are anticipated to be useful in treating diabetes are compounds that bind to the AMP site of fructose 1,6-bisphosphatase (FBPase) as described by U.S. Pat. No. 5,658,889, WO 98/39344, WO 98/39343, and WO 98/39342. Other examples of phosphorus-containing drugs include squalene synthetase inhibitors (e.g. BMS 188494).
A large class of drugs known to be active against hepatitis are generally nucleoside or nucleotide analogs that are phosphorylated inside cells to produce the biologically active triphosphate. Examples include Lamivudine (3TC) and Vidarabine (araA). In each case, the drug interferes with viral replication via the triphosphate form through either inhibition of the viral DNA polymerases or DNA chain termination. Some specificity for virus-infected cells is gained by both preferential phosphorylation of the drug by virally-encoded kinases as well as by specific inhibition of viral DNA polymerases. Nevertheless many of the nucleoside-based drugs are associated with significant non-hepatic toxicity. For example, araA frequently produces neurological toxicity (40%) with many patients showing myalgia or a sensory neuropathy with distressing pain and abnormalities in nerve conduction and a few showing tremor, dysarthria, confusion or even coma. Lok et al., J. Antimicrob. Chemotherap. 14: 93-99 (1984).
Phosphonic acids also show antiviral activity. In some cases the compounds are antivirals themselves (e.g. phosphonoformic acid), whereas in other cases they require phosphorylation to the disphosphate, e.g. 9-(2-phosphonylmethoxyethyl)adenine (PMEA, Adefovir). Frequently, these compounds are reported to exhibit enhanced activity due to either poor substrate activity of the corresponding nucleoside with viral kinases or because the viral nucleoside kinase which is required to convert the nucleoside to the monophosphate is down regulated viral resistance. Monophosphates and phosphonic acids, however, are difficult to deliver to virally-infected cells after oral administration due to their high charge and in the case of the monophosphate instability in plasma. In addition, these compounds often have short half-lives (e.g. PMEA, Adefovir) due in most cases to high renal clearance. In some cases, the high renal clearance can lead to nephrotoxicities or be a major limitation in diseases such as diabetes where renal function is often compromised.
Liver cancer is poorly treated with current therapies. In general, liver tumors are resistant to radiotherapy, respond poorly to chemotherapy and are characterized by a high degree of cell heterogeneity. Similar compounds as those described for hepatitis are also compounds that are useful for cancer (e.g. 2-Fluoroarabinosyladenosine (F-ara-A, Fludarabine), 2′2′-difluorodeoxycytidine (dFdC, Gemcitabine) and 5-fluorouracil or 5-fluoro-2′-deoxy uridine.
Hepatitis and liver cancer remain poorly treated with current therapies due to dose-limiting extrahepatic side effects or inadequate delivery of chemotherapeutic agents to the target tissue. Efforts to deliver drugs to the liver with relatively high organ specificity have primarily focused on strategies involving receptor mediated endocytosis (RME). RME transport systems are common to normal macrophages, hepatocytes, fibroblasts and reticulocytes. Macromolecules internalized via RME include asialoglycoproteins, LDL, transferrin and insulin. Another strategy for drug delivery to the liver uses colloids or liposomes both of which are subject to phagocytosis by the macrophage (Kupffer cells in liver) and localization in tissues of the reticuloendothelial system (e.g. liver, spleen and bone). Of these possible approaches, most of the attention has focused on the use of glycoprotein and oligosaccharide drug conjugates as a method for organ specific delivery. Natural desialylated glycoproteins, e.g. asialoorosomucoid and asialofetuin, neoglycoproteins, e.g. mannosylated and lactosylated albumin, and polysacharrides such as arabinogalactan have been used to successfully deliver drugs to the liver.
Conjugates of several drug classes have been reported, including the antiviral drug araAMP. For example, araAMP conjugated to lactosaminated serum albumin was effective in treating chronic type B hepatitis without signs of neurotoxicity. Fiume et al., The Lancet 13 (1988). Because conjugation of drugs to plasma proteins may have several limitations, including uptake by scavenger receptors on non-hepatocytes, immunogenicity and instability of the protein to conjugation conditions, and in vivo metabolism, efforts have focused on the use of oligosaccharide conjugates. One such approach uses arabinogalactan conjugate. The araAMP conjugate is reported to have good activity in woodchucks carrying the hepatitis virus. Enriquez et al., Bioconj. Chem. 6: 195-202 (1995).
Limitations in approaches described above include drug loading capacity, complexity of the manufacture and characterization of the conjugate, and receptor down regulation. Thus, there is still a need for prodrugs of phosphorus containing drugs.